Method and apparatus for detecting and counting platelets individually and in aggregate clumps

ABSTRACT

A method for enumerating platelets within a blood sample is provided. The method includes the steps of: 1) depositing the sample into a sample container having an analysis chamber adapted to quiescently hold the sample for analysis, and an amount of colorant that platelets absorb and which fluoresces upon exposure to one or more predetermined first wavelengths of light; 2) imaging at least a portion of the sample disposed in the analysis chamber, including producing image signals indicative of fluorescent emissions from the platelets illuminated by first wavelengths of light; 3) identifying the platelets using the image signals; and 4) enumerating individual platelets and clumped platelets within the sample using one or more of fluorescent emissions, area, shape, and granularity.

The present application is a continuation of U.S. patent applicationSer. No. 13/088,853 filed Apr. 18, 2011, which is a continuation of U.S.Pat. No. 7,929,121 filed Mar. 20, 2009, which claims priority to U.S.Provisional Patent Appln. No. 61/038,554, filed Mar. 21, 2008.

BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to apparatus and methods for analysis ofblood samples in general, and apparatus and methods for detecting andenumerating platelets, and differentiating platelets from giantplatelets, and giant platelets from platelet clumps, in particular.

2. Background Information

Physicians, veterinarians and scientists have examined human andanimals' biologic fluids, especially blood, in order to determineconstituent quantities as well as to identify the presence of unusualparticulates not seen in healthy subjects. The constituents generallymeasured, quantified and identified include red blood cells (RBCS),white blood cells (WBCs), and platelets.

In mammals, platelets (also referred to as thrombocytes) are smallirregularly shaped anuclear cell fragments that are derived fromfragmentation of megakaryocytes. Thrombocytes in certain animals (e.g.,birds, reptiles and fish) are similar in function to mammalianplatelets, but are about ten times larger and nucleated. Plateletanalyses can include the number, size, shape, texture, and volumedeterminations of the platelets within the sample, including thedetermination of the presence of clumps of platelets or thrombocyteswithin the sample. Under certain naturally occurring conditions,platelets will aggregate into clumps within a subject as a usefulresponse to a trauma (e.g., hemorrhaging, tissue trauma, etc.)experienced by the body. Platelet clumps forming within a blood samplecollected for analysis, on the other hand, are typically not useful andcan hinder the analysis of the blood sample. Anticoagulants (e.g., EDTA)can be used to prevent platelets from clumping within a sample, butclumps may still form if there is delay in mixing the anticoagulant withthe blood sample. Once clumps form, anticoagulants are typicallyineffective in separating them into individual platelets. Plateletclumps are often problematic within a sample being analyzed because theycan lead to erroneously low platelet counts, which can lead tomisdiagnosis and serious consequences to the patient.

Known blood examination techniques, described in detail medical textssuch as Wintrobe's Clinical Hematology 12^(th) Edition, generally dividethe examination methods into manual, centrifugal, and impedance typemethods. Manual methods typically involve the creation of an accuratelydetermined volume of a blood or fluid sample that is quantitativelydiluted and visually counted in a counting chamber. Manual examinationmethods for cell enumeration include examining a peripheral smear wherethe relative amounts of the particulate types are determined by visualinspection. Centrifugal examination methods involve centrifuging thesample, causing the sample to separate into constituent layers accordingto the relative densities of the constituents. The component layers canbe stained to enhance visibility or detection. Impedance methods involvethe examination of an accurate volume of blood which is treatedaccording to the particulate being measured; e.g., lysing RBCs forenumeration of the nucleated cells and volumetrically diluting thesample in a conductive fluid. The process typically involves monitoringa current or voltage applied to sample passing through a narrow passageto determine the effect particulates have on the current/voltage as theparticulates pass through in single file. Other techniques involveanalyzing the intensity and angle of scatter of light incident toparticulates passing single file through a light beam. Flow cytometricmethods can also be used that involve staining particulates of interestin suspension with fluorophores attached to antibodies directed againstsurface epitopes present on cell or particle types, exciting the stainedparticulates with light of appropriate wavelengths, and analyzing theemission of the individual particulates/cells.

All of the aforementioned methods, other than the peripheral smear orcentrifugal separation, require dispensing a precise volume of sample.Inaccuracies in the sample volume will result in quantitative errors ofthe same magnitude in the associated analysis. With the exception ofcentrifugal methods, all of the aforementioned methods also require thesample to be mixed with one or more liquid reagents or diluents, andalso require calibration of the instrument to obtain accurate results.In the case of peripheral smears, a high degree of training is needed toproperly examine the smear. A number of the aforementioned methodsgenerate large volumes of contaminated waste which is expensive tohandle. Additionally, the above-described methods are not suitable todetermine the complete blood count (CBC) in birds, reptiles and fish,where the red blood cells and thrombocytes are nucleated, and in certainmammals where the red blood cells size is very small and may be confusedwith platelets.

SUMMARY OF THE INVENTION

According to an aspect of the present invention, a method forenumerating platelets within a substantially undiluted blood sample isprovided. The method includes the steps of: 1) depositing the sampleinto an analysis chamber adapted to quiescently hold the sample foranalysis, the chamber defined by a first panel and a second panel, bothof which panels are transparent; 2) admixing a colorant with the sample,which colorant is operative to cause the platelets to fluoresce uponexposure to one or more predetermined first wavelengths of light; 3)illuminating at least a portion of the sample containing the plateletsat the first wavelengths; 4) imaging the at least a portion of thesample, including producing image signals indicative of fluorescentemissions from the platelets, which fluorescent emissions have anintensity; 5) identifying the platelets by their fluorescent emissions,using the image signals; 6) determining an average fluorescent emissionintensity value for the individual platelets identified within the atleast a portion of the sample; 7) identifying clumps of platelets withinthe at least a portion of the sample using one or more of theirfluorescent emissions, area, shape, and granularity; and 8) enumeratingplatelets within each platelet clump using the average fluorescentemission intensity value determined for the individual platelets withinthe sample.

An advantage of the present invention is that it provides an accurateplatelet count within a blood sample. Most prior art hematologyanalyzers count the number of platelets within the sample by assumingthat constituents within the sample of a certain size are in factplatelets. Giant platelets and platelet clumps, both of which are biggerthan normal size platelets, may not therefore be considered in the countand may be counted as white blood cells. The resultant lower plateletcount can be erroneously interpreted as a thrombocytopenia. The presentinvention identifies giant platelets and platelet clumps and enumeratesthe platelets within the platelet clumps. As a result, a platelet countis provided that is more accurate than that provided by most prior artautomated hematology analyzers and one which avoids counting giantplatelets and platelet clumps as white blood cells resulting in falselylow platelet counts and falsely high white blood cell counts.

Another advantage of the present invention is that it permits theidentification and enumeration of giant platelets within a blood sample.

Another advantage of the present invention is that it can be used todetermine characteristics of a blood sample using an extremely smallsample volume that may be obtained directly from the patient bycapillary puncture rendering it more useful for point of careapplications or from a venous sample if desired.

Another advantage of the present method is that it operates free ofexternal and internal fluidics, and independent of gravity ororientation, and therefore is adaptable for use in a portable or handheld device and in microgravity conditions.

The present method and advantages associated therewith will become morereadily apparent in view of the detailed description provided below,including the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The application file contains at least one drawing executed in color.Copies of this patent or patent application publication with colordrawing(s) will be provided by the Office upon request and payment ofthe necessary fee.

FIGS. 1-4 are cross-sectional diagrammatic representations of analysischambers that may be used in the present method.

FIG. 5 is a diagrammatic planar view of a tape having a plurality ofanalysis chambers.

FIG. 6 is a diagrammatic planar view of a disposable container having ananalysis chamber.

FIG. 7 is a diagrammatic cross-sectional view of a disposable containerhaving an analysis chamber.

FIG. 8 is a diagrammatic schematic of an analysis device that may beused with the present method.

FIG. 9 is a color image of a portion of a chamber containing asubstantially undiluted blood sample admixed with a acridine orangefluorescent dye, that has been illuminated at a wavelength that producesfluorescent emissions from constituents (e.g., WBCs, platelets, etc)that have absorbed the dye, imaged at a first intensity amplification.

FIG. 10 is the color image shown in FIG. 9, imaged at a second intensityamplification that is greater than the first.

FIG. 11 is a color image of a portion of a chamber containing asubstantially undiluted blood sample admixed with a acridine orangefluorescent dye, that has been illuminated at a wavelength that producesfluorescent emissions from constituents, illustrating the differentemission profiles of WBCs, platelet clumps, and platelets.

FIG. 12 is a color image of a portion of a chamber containing asubstantially undiluted blood sample admixed with a acridine orangefluorescent dye, that has been illuminated at a wavelength that producesfluorescent emissions from constituents, illustrating the differentemission profiles of platelets, giant platelets, and reticulocytes.

FIG. 13 is a color image of a portion of a chamber containing asubstantially undiluted blood sample admixed with a acridine orangefluorescent dye, that has been illuminated at a wavelength that producesfluorescent emissions from constituents, illustrating the differentemission profiles of platelets and reticulocytes. The image alsoincludes WBCs edited from the image, and RBCs faintly seen in thebackground.

FIG. 14 is a black and white version of the image shown in FIG. 13 whichis created by illuminating the sample at a wavelength that shows theoptical density values of the image. This image illustrates the opticaldensity profiles of reticulocytes and RBCs within the sample, whichprofiles enables reticulocytes to be identified.

FIG. 15 is a block diagram of the steps a method according to an aspectof the present invention.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The present method generally utilizes an analysis chamber that isoperable to quiescently hold a sample of substantially undilutedanticoagulated whole blood for analysis. The chamber is typically sizedto hold about 0.2 to 1.0 μA of sample, but the chamber is not limited toany particular volume capacity, and the capacity can vary to suit theanalysis application. The phrase “substantially undiluted” as usedherein describes a blood sample which is either not diluted at all orhas not been diluted purposefully, but has had some reagents addedthereto for purposes of the analysis. To the extent the addition of thereagents dilutes the sample, if at all, such dilution has no clinicallysignificant impact on the analysis performed. Typically, the onlyreagents that will be used in performing the present method areanticoagulants (e.g., EDTA, heparin) and colorants. These reagents aregenerally added in dried form and are not intended to dilute the sample.Under certain circumstances (e.g., very rapid analysis—such as mayhappen when blood is drawn from a patient finger stick or a neonatalheel-stick), it may not be necessary to add the anticoagulating agent,but it is preferable to do so in most cases to ensure the sample is in aform acceptable for analysis. The term “quiescent” is used to describethat the sample is deposited within the chamber for analysis, and is notpurposefully moved relative to the chamber during the analysis. To theextent that motion is present within the blood sample, it willpredominantly be that due to Brownian motion of the blood sample'sformed constituents, which motion is not disabling of the use of thedevice of this invention.

The colorant (e.g., a dye, stain, etc.), which is admixed with at leasta portion of the blood sample, facilitates identification andquantitative analysis of the constituents (e.g., platelets, WBCs, etc.)that absorb the colorant. The colorant fluoresces along characteristicwavelengths (e.g., 530 nm, 585 nm, and 660 nm) when excited by lightalong certain wavelengths (e.g., about 470 nm). The specific wavelengthsat which a constituent will fluoresce are a characteristic of thatconstituent and the wavelength(s) of the exciting light. In someembodiments, the colorant can also absorb light at one or morepredetermined wavelengths as a function of the concentration of thecolorant within the constituent. Examples of acceptable colorantsinclude the supravital dyes acridine orange and astrozone orange. Theinvention is not limited to supravital dyes, however. A person of skillin the art would know appropriate concentration ranges of colorants, orcould determine the same without undue experimentation.

Now referring to FIG. 1, the analysis chamber 10 is defined by a firstpanel 12 having an interior surface 14, and a second panel 16 having aninterior surface 18. The panels 12, 16 are both sufficiently transparentto allow the transmission of light along predetermined wavelengths therethrough in an amount sufficient to perform the optical density analysisdescribed below. At least a portion of the panels 12, 16 are parallelwith one another, and within that portion the interior surfaces 14, 18are separated from one another by a height 20, which height may be knownor measurable. RBCs 22 are shown disposed within the chamber 10.

The present method can utilize a variety of different analysis chamberstypes having the aforesaid characteristics, and is not therefore limitedto any particular type of analysis chamber. An analysis chamber havingparallel panels 12, 16 simplifies the analysis and is thereforepreferred, but is not required for the present invention; e.g., achamber having one panel disposed at a known non-parallel angle relativeto the other panel could be used.

Now referring to FIGS. 2-5, an example of an acceptable chamber 10 isshown that includes a first panel 12, a second panel 16, and at leastthree separators 26 disposed between the panels 12, 16. The separators26 can be any structure that is disposable between the panels 12, 16,operable to space the panels 12, 16 apart from one another. Thedimension 28 of a separator 26 that extends between the panels 12, 16 isreferred to herein as the height 28 of the separator 26. The heights 28of the separators 26 typically do not equal one another exactly (e.g.,manufacturing tolerances), but are within commercially acceptabletolerance for spacing means used in similar analysis apparatus.Spherical beads are an example of an acceptable separator 26 and arecommercially available from, for example, Bangs Laboratories of Fishers,Ind., U.S.A.

In the chamber embodiment shown in FIG. 3, the separators 26 consist ofa material that has greater flexibility than one or both of the firstpanel 12 and the second panel 16. As can be seen in FIG. 3, the largerseparators 26 are compressed to the point where most separators 26 aretouching the interior surfaces of the panels 12, 16, thereby making thechamber height just slightly less than the mean separator 26 diameters.In the chamber embodiment shown in FIG. 4, the separators 26 consist ofa material that has less flexibility than one or both of the first panel12 and the second panel 16. In FIG. 4, the first panel 12 is formed froma material more flexible than the spherical separators 26 and the secondpanel 16, and will overlay the separators 26 in a tent-like fashion. Inthis embodiment, although small local regions of the chamber 10 maydeviate from the desired chamber height 20, the average height 20 of thechamber 10 will be very close to that of the mean separator 26 diameter.Analysis indicates that the mean chamber height 20 can be controlled toone percent (1%) or better at chamber heights of less than four micronsusing this embodiment. Subject to the flexibility characteristicsdescribed above (as well as other factors such as the distributiondensity of the separators), the separators 26 and panels 12, 16 can bemade from a variety of materials, provided the panels 12, 16 aresufficiently transparent. Transparent plastic films consisting ofPolyethylene terephthalate is an example of acceptable panels 12, 16,and spherical beads made of polystyrene, polycarbonate, silicone, andthe like, are acceptable separators 26. A specific example of anacceptable separator is spheres made of polystyrene that arecommercially available, for example, from Thermo Scientific of Fremont,Calif., U.S.A., catalogue no. 4204A, in four micron (4 μm) diameter.Referring to FIG. 5, the panel 12 that is to be vertically disposedabove the other includes a plurality of ports 30 disposed at regularintervals (e.g., that act as air vents), and the panels 12, 16 arebonded together at points. In some embodiments, the bonding material 32forms an outer chamber wall operable to laterally contain the sample 34within the analysis chamber 10. This example of an acceptable analysischamber is described in greater detail in U.S. Patent ApplicationPublication Nos. 2007/0243117, 2007/0087442, and U.S. Provisional PatentApplication Nos. 61/041,783, filed Apr. 2, 2008; and 61/110,341, filedOct. 31, 2008, all of which are hereby incorporated by reference intheir entirety.

Another example of an acceptable chamber 10 is disposed in a disposablecontainer 36 as shown in FIGS. 6 and 7. The chamber 10 is formed betweena first panel 12 and a second panel 16. Both the first panel 12 and thesecond panel 16 are transparent to allow light to pass through thechamber 10. At least a portion of the first panel 12 and the secondpanel 16 are parallel with one another, and within that portion theinterior surfaces 14, 18 are separated from one another by a height 20.This chamber 10 embodiment is described in greater detail in U.S. Pat.No. 6,723,290, which patent is hereby incorporated by reference in itsentirety. The analysis chambers shown in FIGS. 2-7, represent chambersthat are acceptable for use in the present method. The present method isnot, however, limited to these particular embodiments.

It is not necessary to know the exact height of the chamber for purposesof the present disclosure. A chamber height of about two to six microns(2-6μ) is acceptable for most animal species based on typical cellsizes. A chamber height 20 of about three to five microns (3-5μ) isparticularly well suited for analyzing human blood. The presentinvention is not, however, limited to any particular chamber heightprovided the methodology described herein can be accomplished with suchchamber height.

The analysis of the sample quiescently disposed within the chamber 10 isperformed using an analysis device that is operable to illuminate andimage at least a portion of the sample and perform an analysis on theimage. The image is produced in a manner that permits fluorescentemissions from, and the optical density of, the portion of the sample tobe determined on a per unit basis. The term “per unit basis” or “imageunit” means a defined incremental unit of which the image of the samplecan be dissected. A pixel, which is generally defined as the smallestelement of an image that can be individually processed within aparticular imaging system, is an example of an image unit, and an imageunit may include a small number of pixels in a collective unit. Themagnification of an imaging device can also be described in linear terms(e.g., microns per pixel at the focal plane), where the linear dimensionis along a particular axis of an orthogonal grid applied to the image.The actual area of the sample captured by pixels of the sensor at thefocal plane is therefore a function of the magnification factor appliedby the imaging device. Hence, it is useful but not required to know themagnification of the imaging device. The volume associated with thatpixel is therefore the area of the image per pixel times the chamberheight. For example if the magnification was 0.5 microns per pixel, animage occupying 200 pixels would have an area of 50 square microns, anda volume of 50 square microns times the chamber height.

Now referring to FIG. 8, an example of an analysis device 44 that can beadapted for use with the present method includes a sample illuminator46, an image dissector 48, and a programmable analyzer 50. The sampleilluminator 46 includes a light source that selectively produces lightalong certain desired wavelengths. For example, LEDs that emit thedesired wavelengths (e.g., 420 nm, 440 nm, 470 nm, etc.) can be used.Alternatively, a light source that produces a broad wavelength range(e.g., approximately 400-670 nm) can be used, although in some instancessuch a light source may require filtering. The analysis device 44 mayinclude optics for manipulating the light. The sample illuminator 46includes a transmittance light source and an epi-illumination lightsource, each operable to illuminate some, or all, of the sample residingwithin the chamber 10. An example of an acceptable image dissector 48 isa charge couple device (CCD) type image sensor that converts an image ofthe light passing through the sample into an electronic data format(i.e., a signal). Complementary metal oxide semiconductor (“CMOS”) typeimage sensors are another example of an image sensor that can be used,and the present invention is not limited to either of these examples.The programmable analyzer 50 includes a central processing unit (CPU)and is connected to the sample illuminator 46 and image dissector 48.The CPU is adapted (e.g., programmed) to selectively perform thefunctions necessary to perform the present method. It should be notedthat the functionality of programmable analyzer 50 may be implementedusing hardware, software, firmware, or a combination thereof. A personskilled in the art would be able to program the processing unit toperform the functionality described herein without undueexperimentation. U.S. Pat. No. 6,866,823 entitled “Apparatus forAnalyzing Biologic Fluids” and issued Mar. 15, 2005, which is herebyincorporated by reference in its entirety, discloses such an analysisdevice 44.

The analysis device is adapted to process the image signals created fromthe illumination of at least a portion of the sample to identify andenumerate constituents within the sample. The image signals includefluorescent emissions and optical density values on a per pixel basis.The intensity and color of the emissions and the optical density perpixel collectively establish the image of the illuminated sampleportion. Within the collective image, the analysis device is adapted toidentify a profile of selected constituents, using one or more of thefluorescent intensity, color content, and optical density of thefluorescent emissions, and in some instances physical characteristics(e.g., area, edge geometry, etc.) of the constituents. The analysisdevice uses the image profiles to distinguish amongst the selectedconstituents until the remaining constituents represent the targetconstituents (e.g., platelets), at which point the target constituentscan be enumerated.

Under the present method, a sample of substantially undiluted wholeblood is introduced into a chamber 10, and thereinafter residesquiescently within the chamber 10. An anticoagulating agent and acolorant are admixed with the sample either prior to its introductioninto the chamber or upon introduction into the chamber. The colorant isabsorbed by the constituents (e.g., WBCs, platelets, reticulocytes)within the sample. In some applications, an isovolumetric sphering agentis added to the sample to cause some or all of the RBCs within thesample to assume a spherical-like shape. An example of an acceptableisovolumetric sphering agent is a zwitterionic detergent. A specificexample of a sphering agent is Zwittergent® 3-16 detergent, which is azwitterionic detergent produced by Calibriochem, an entity of EMDChemicals, Inc. of New Jersey, U.S.A. The amount of sphering agent addedto the sample is an amount adequate to sphere at least a number of RBCsrequired to perform the present hematocrit analysis. The specific amountwill depend on the particular agent and test circumstances, which can bedetermined by a person of skill in the art without undueexperimentation. The natural bioconcave shape of RBCs, and the relativesize of RBCs relative to platelets, can cause platelets within a sampleto be “hidden” amongst the RBCs in the sample; e.g., within theconcavities of an RBC. Sphering the RBCs decreases the likelihood ofplatelets being hidden within a sample amongst the RBCs and increasesthe likelihood that such platelets can be viewed as individuals withinthe plasma, and thereby increases the accuracy of quantitative plateletanalyses performed on the sample.

At least a portion of the sample quiescently residing within the chamberis illuminated by the analysis device 44, which transmits light throughthe sample. Although it is not a requirement that the entire sampleresiding within the chamber be imaged, it is preferable since doing sotypically provides a more complete analysis of the sample and aconcomitant increase in accuracy. The sample is illuminated withwavelengths known to excite a fluorescent emission from the constituentsrelating to the colorant absorbed by the constituents. Constituentsstained with acridine orange produce a fluorescent emission whenilluminated with violet light at a wavelength of about 470 nm. Thephotographs shown in FIGS. 9-13 illustrate the fluorescent emissions ofconstituents (e.g., platelets, giant platelets, WBCs, reticulocytes,platelet clumps) found within the sample. The specific emissions dependupon the colorant used and the intracellular composition of theilluminated cell (e.g., interaction of the colorant with contents of thecell or platelet creates the emissions). Some constituents havefluorescent emissions that act as a fluorometric signature (alsoreferred to as a “profile”), which signature represents a particularratio of fluorescent emissions along wavelengths that producecombinations of light (e.g., a characteristic “red/green” ratio) thatare relatively unique to that constituent and can therefore be used toidentify that constituent. Other constituents have fluorescent emissionsignatures that cannot easily be distinguished from one another. Todistinguish those constituents, the sample is illuminated withwavelengths of light that are absorbed by hemoglobin in appreciablygreater amounts than would be absorbed by the platelets or plasma. Theamount of absorption can be measured in terms of optical density, andthe optical density in turn can be used to distinguish constituentscontaining hemoglobin from those that do not.

Because the fluorescent emission portion of the image is a function offactors such as the type of colorant used and the concentration of thecolorant within the sample, it is useful, but not required, to calibratethe sample for intensity. For example, for a given concentration ofcolorant, the fluorescent emission from WBCs is on average higher thanthe fluorescent emission from platelets. This can be clearly seen inFIGS. 9 and 10, which are images of the same sample. The amplificationof the fluorescent emission in FIG. 10 is greater than that used in FIG.9. FIG. 9 clearly shows the fluorescent emissions from the WBCs 40, andfaintly shows the emissions from the platelets 42. FIG. 10 clearly showsboth WBCs 40 and platelets 42, and also shows how each can bedistinguished by their fluorescent emission intensity. The calibrationidentifies the intensity level associated with the WBCs and theintensity level associated with the platelets to the analysis device,and calibrates the analysis device accordingly.

The fluorescent emissions and transmitted light produced by illuminatingthe sample are converted into image signals on a per pixel basis thatcollectively establish the image of the illuminated sample portion.Within the collective image, the analysis device is adapted to identifya profile of certain selected constituents, using one or more of thefluorescent intensity, color content, and optical density of thefluorescent emissions. The process of identifying the constituentsprofiles via aforesaid characteristics can be performed using algorithmsthat compare the various characteristics to identify the constituentOnce the constituent is identified, it can be further analyzed. Forexample, a representative number of platelets can be identified withinthe sample via their fluorescent emission profile and collectivelyanalyzed to determine an average of the fluorescent emission intensityfor the platelets. In some instances, constituent fluorescent profilesare also used to determine the internal areas and edge regions of theconstituents using the fluorescent signal profiles. The areas ofindividual constituents can be averaged to determine an average areavalue. The edge profiles can be analyzed for smoothness and/or forgeometry; e.g., determine if the edge of a constituent is circular,non-circular, irregular, etc. These characteristics are subsequentlyused to distinguish constituents within the sample, until the remainingconstituents represent the target constituents (e.g., platelets), atwhich point the target constituents can be enumerated.

To illustrate an example of the present invention, a substantiallyundiluted sample of blood is admixed with EDTA, acridine orange, and azwitterionic detergent and is introduced within a chamber having twotransparent panels for the purpose of determining a platelet countwithin the sample. Constituents, including RBCs, reticulocytes, WBCs,platelets, giant platelets, and platelet clumps, reside quiescentlywithin the sample. The sample is illuminated at 470 nm, at least one of413 nm and 540 nm. The 470 nm illumination produces a fluorescentemission from the acridine orange. Other colorants may emit light uponillumination at other wavelengths. The 413 nm and/or 540 nm illuminationis used to indicate the presence of hemoglobin by its optical density,as will be discussed below. Digital images of the illuminated sample aretaken.

The image of the sample is analyzed to identify various constituentsdisposed within the sample. WBCs, for example, are individuallyidentified by one or more of their fluorescent signature (e.g.,fluorescent emission pattern consisting of a significant red cytoplasmicfluorescence and a green nuclear fluorescence), the relative intensityof their fluorescent emissions, the area they occupy, and their shape(see FIGS. 9-11). The WBCs are thereby distinguished from the remainderof the sample; e.g., by filtering the image so that WBCs are no longerconsidered within the image.

Now referring to FIG. 12, giant platelets 44 can be identified withinthe sample by one or more of their fluorescent profile, area, and shape.RBCs 45 can be seen faintly in the background. The colorimetric ratio ofa giant platelet 44 is similar to that of a normal platelet 42, but theemissions are greater in intensity due to the larger mass of theparticle. Giant platelets 44 are also significantly larger than normalplatelets 42 and circular in size. Normal platelets 42 are typicallyirregularly shaped. Most normal sized platelets are 1.5 to 3 μm indiameter. Giant platelets 44, in contrast, are larger than 7 μm andusually in the range of 10 μm to 20 μm in diameter. The identificationand enumeration of giant platelets provides important clinicalinformation, since the presence of giant platelets may be an indicatorof Bernard-Soulier syndrome and myeloproliferative disorders (e.g.,chronic myelogenous leukemia (CML), polycythemia vera, essential(primary) thrombocythemia, and agnogenic myeloid metaplasia). Giantplatelets 44 within the sample are identified by comparing one or moreof their fluorescent emissions (colorimetric and intensity), area, andperimeter shape against normal platelet fluorescent emission values,area, and perimeter shapes, including the average platelet intensity andarea values. As an example of criteria that can be used to identifygiant platelets, the analysis device can be programmed with one or morecomparative criteria relative to normal platelets (e.g., a multiple ofthe average platelet area or intensity based on standard deviations ofthe average normal platelet area or intensity, or by a predeterminedarea or intensity value, etc.). With respect to with average plateletarea, the distribution of platelet areas within any given sample istypically described as a log-normal function and can be determinedstatistically using known techniques. The identified giant platelets arethen distinguished and, depending upon the specific information desired,are included in the platelet count and/or are considered as anindependent constituent population.

Reticulocytes 46 emit a fluorescent profile that is similar to that ofnormal platelets because of the nuclear material they contain. Thephotograph in FIG. 13 shows the fluorescent emissions of reticulocytes46 and platelets 42. The circular blacked out portions are portions ofthe image where WBCs were disposed, but which image was masked out. Thereticulocytes 46 can be distinguished from platelets 42 to some extentby their fluorescent emission, which appears slightly brighter and hasslightly more red. The reticulocytes 46 can also be distinguished byilluminating the sample with light at wavelengths of 413 nm and/or 540nm, which wavelengths are absorbed by the hemoglobin to a substantiallygreater amount than any other material present within the sample and is,therefore, indicative of the presence of hemoglobin. The lightabsorption of the hemoglobin can be quantified in terms of opticaldensity. FIG. 14 illustrates the OD of the reticulocytes. Thereticulocytes are distinguished from the remainder of the sample usingone or both of the fluorescent emission patterns and OD.

In most blood samples, the constituents with a fluorescent emissionremaining after the WBCs, giant platelets, and reticulocytes have beendistinguished are predominantly, if not entirely, platelets. Individualplatelets within the sample can be identified and enumerated. In someblood samples, however, a portion of the platelets within the sample maybe aggregated into one or more clumps, which clumps can be very large insize; e.g., one to four times the size of a WBC. The photograph in FIG.11 shows both platelet clumps 48 and WBCs 40. The platelet clumps 48 areidentifiable and distinguishable from other constituents (e.g., WBCs 40,giant platelets 44) by their fluorescent emission profile which has ared/green ratio that is distinguishable from that of a WBC 40, by theirgranularity, and in some instances by their area and/or shape. Forexample, platelet clumps 48 are distinguishable from giant plateletsbecause platelet clumps are irregular shaped and giant platelets aresubstantially circularly shaped. The relative circularity of bothplatelet clumps and giant platelets can be determined by image analysissoftware programmed into analysis device. Detecting the presence ofplatelet clumps within the sample is important because of thepossibility that the clumps will be counted as WBCs (and thereby falselyelevate the WBC count) and/or lead to a false diagnosis ofthrombocytopenia. These potential problems are particularly relevantwith existing automated analyzers. The presence of platelet clumps canbe an indicator of inadequate mixing of the sample with EDTA.

Once a platelet clump is identified, the integrated fluorescent emissionintensity of the clump and the area of the clump can be determined. Thenumber of platelets within the clump can then be determined by dividingthe integrated fluorescent emission intensity by the average plateletemission intensity. The quotient value is an acceptable approximation ofthe actual number of platelets within the clump. An approximation of thenumber of platelets within a clump can also be determined by dividingthe area of the clump by the average platelet area.

Although this invention has been shown and described with respect to thedetailed embodiments thereof, it will be understood by those skilled inthe art that various changes in form and detail may be made withoutdeparting from the spirit and scope of the invention.

1. A method for enumerating platelets within a blood sample, comprisingthe steps of: depositing the sample into a sample container having ananalysis chamber adapted to quiescently hold the sample for analysis,and an amount of colorant that platelets absorb and which fluorescesupon exposure to one or more predetermined first wavelengths of light;imaging at least a portion of the sample disposed in the analysischamber, including producing image signals indicative of fluorescentemissions from the platelets illuminated by first wavelengths of light;identifying the platelets using the image signals, and one or more ofarea, shape, and granularity characteristics; and enumerating individualplatelets within the sample.
 2. The method of claim 1, wherein the stepof identifying the platelets includes identifying giant platelets withinthe sample.
 3. The method of claim 2, wherein the step of identifyingthe platelets includes determining an average area for the individualplatelets identified within the sample, and comparing a determined areaof at least one giant platelet identified within the sample to theaverage platelet area.
 4. The method of claim 2, wherein the step ofidentifying giant platelets within the sample includes comparing thearea of at least one giant platelet to a predetermined value.
 5. Amethod for enumerating platelets within a blood sample, which sample isdisposed in an analysis chamber adapted to quiescently hold the samplefor analysis, and which sample includes an amount of colorant thatplatelets absorb and which fluoresces upon exposure to one or morepredetermined first wavelengths of light, the method comprising thesteps of: imaging at least a portion of the sample disposed in theanalysis chamber, including producing image signals indicative offluorescent emissions from the platelets illuminated by firstwavelengths of light; identifying the platelets using the image signals,and one or more of area, shape, and granularity characteristics; andenumerating platelets within the sample.
 6. The method of claim 5,wherein the platelets enumerated within the sample include one or moreof individual platelets, clumped platelets and giant platelets.
 7. Anapparatus for enumerating platelets within a blood sample, which sampleincludes a colorant operative to cause the platelets to fluoresce uponexposure to one or more predetermined first wavelengths of light, theapparatus comprising: an analysis chamber adapted to quiescently holdthe sample for analysis, the chamber defined by a first panel and asecond panel; an imaging unit that is operable to image at least aportion of the sample and produce image signals indicative offluorescent emissions from the platelets; and a programmable analyzeradapted to identify and enumerate platelets within the sample, using theimage signals, and area, shape, and granularity characteristics.
 8. Theapparatus of claim 7, wherein the programmable analyzer is furtheradapted to determine a total number of platelets within the sample as asum of platelets within one or more platelet clumps and plateletsindividually residing within the sample.
 9. The apparatus of claim 7,wherein the programmable analyzer is adapted to identify giant plateletswithin the sample.
 10. The apparatus of claim 9 wherein the programmableanalyzer is adapted to identify giant platelets within the sample by oneor more of fluorescent emissions, area, and shape.
 11. The apparatus ofclaim 7, wherein the imaging unit is operable to image at least aportion of the sample and produce second image signals indicative of anoptical density of one or more constituents within the sample; andwherein the programmable analyzer is adapted to determine the opticaldensity of at least one of the constituents within the sample using thesecond image signals, and to distinguish the platelets and theconstituents from one another using the determined optical density.